This proposal is designed to test a novel way to activate T lymphocytes toward weak antigens and to develop a means to deliver this novel activator into animals (and ultimately, humans) for treating established cancers. A fundamental mechanism regulating immune tolerance is the selection of the T cell repertoire, eliminating T cells expressing receptors with strong reactivity against self. However, there still exist T cells expressing receptors with weak reactivity, not capable of being activated by weak antigens (as exist on tumors). It is well known that once these nave T cells receive a robust activation signal they can become competent killer cells when they subsequently encounter tumors. Our experimental design is based on detailed structural knowledge honed over more than 30 years of studies that defined differences between receptor-ligand interactions in the tolerant and activation states. We propose to make structural changes in conserved amino acids at the interface of Major Histocompatibility Complex (MHC)-encoded molecules in regions that interact with germ-line encoded parts of T cell receptors (TcRs), testing these altered MHCs for enhanced TcR binding and robust T cell activation signals, while maintaining the nature of receptor-ligand interactions which define antigen specificity. In this high risk/high reward proposal, we will (1) test the hypothesis that MHC ligands can be generated with the property of activating normally tolerant T cells with autoreactive properties, in vitro and in vivo, and (2) develop and test a viral vector capable of delivering the antigen-specific stimulatory signal in vivo. Support from the R21 mechanism is essential for addressing these critical questions and will provide needed information that will be foundational for future studies using preclinical models and the subsequent translation using human HLA molecules similarly formulated for clinical trials. Maintenance of antigen specificity without needing to personalize each vaccine for a given patient or tumor type allows this platform to thereby be an off the shelf therapy to benefit a wide variety of cancer patients.